Three new prenylflavonol glycosides from heat-processed Epimediumkoreanum

1 Introduction

Herba Epimedii (Yinyanghuo) is one of the most well-known and frequently used Chinese herbal medicine with tonic, anti-rheumatic, and aphrodisiac effects. The genus Epimedium (Berberidaceae) is widely distributed in China, with about 30 species, 15 of which are circulated in the crude drug markets used as Yinyanghuo [1]. The aerial parts of Epimedium koreanum, E. brevicornu, E. sagittatum, and E. pubescens are designated in Chinese Pharmacopoeia (2010 Edition) as the official source of Herba Epimedii [2]. In addition to the aerial parts, the underground parts of Epimedium plants are widely used as anti-rheumatic medicine in Chinese folk medicines [1]. Previous investigations on Epimedium plants have reported prenylflavonoids with various pharmacological effects, such as anti-inflammatory [3], anti-osteoporosis [4–6], antioxidant [7], estrogenic and anti-estrogenic [8], anti-tumor [9], anti-aging activities [10], and so forth. However, the pharmacological activities are different between crude and processed Epimedium. Crude Epimedium is mainly used against rheumatism and in strengthening the bones, whereas processed Epimedium possesses kidney-nourishing, aphrodisiac, and anti-inflammatory effects [11].

As a continuation of our research on Epimedium species [3, 12] and to search for anti-inflammatory prenylflavonoids from processed Epimedium, three new prenylflavonol glycosides, epimedkoresides A–C (1–3), along with 15 known ones, were isolated from the leaves of E. koreanum by heating with sheep fat (Fig. 1). The known compounds were identified as acuminatoside (4) [13], ikarisoside B (5) [14], icariin (6) [15], epimedins A–C (7–9) [16], epimedoside A (10) [15], desmethylicaritin 3-O-β-d-fucopyranosyl (1 → 2)-α-l-rhamnopyranoside-7-O-β-d-glucopyranoside (11) [17], baohuoside V (12) [18], diphylloside B (13) [19], diphylloside A (14) [19], epimedoside E (15) [19], anhydroicaritin 3-O-β-d-fucopyranosyl(1 → 2)-α-l-rhamnopyranoside-7-O-β-d-glucopyranoside (16) [17], hexandraside F (17) [20], and 4′,5-dihydroxyl-8-(3, 3-dimethylallyl)-flavonol-3-O-[β-d-xylopyranosyl(1 → 3)-α- l-rhamnopyranozside]-7-O-β-d-glucopyranoside (18) [21] through comparison of their spectral data with those in the literature. Compounds 5, 7, 9, 12–15 were evaluated for their anti-inflammatory activity. Unfortunately, the results failed to meet expectations. Herein, this article deals with the isolation, structural determination, and biological evaluation of these compounds.

Fig. 1:

Structures of compounds 1–18.

2 Result and discussion

Compound 1 was obtained as a yellow amorphous powder. The positive HR-ESI-MS showed a quasi-molecular ion peak at m/z = 703.2210 ([M + Na]⁺), indicating the molecular formula C₃₂H₄₀O₁₆ and possessing 13 degrees of unsaturation. Its ¹H NMR spectrum (Tables 1 and 2) displayed a pair of broad doublets (δ_H = 7.85 [J = 8.6 Hz] and 6.94 ppm [J = 8.6 Hz]) attributed to the protons of an AA′BB′-type benzene ring, an aromatic singlet (δ_H = 6.24 ppm) attributed to the proton of a penta-substituted aromatic ring, as well as two anomeric protons (δ_H = 4.43 [d, J = 7.6 Hz] and 5.75 ppm [s]) of two sugar residues. Additionally, two sets of two-proton multiplets (δ_H = 2.82 [m, H-11] and 1.68 ppm [m, H-12]) as well as two tertiary methyls (δ_H = 1.26 [s, Me-14] and 1.27 ppm [s, Me-15]) showed a γ-hydroxy-γ,γ-dimethylpropyl group [22, 23], which was further supported by a series of signals at δ_C = 18.8 (t, C-11), 43.9 (t, C-12), 71.5 (s, C-13), 28.9 (q, C-14), and 29.0 ppm (q, C-15) in the ¹³C NMR spectrum (Table 1). Except for 17 sp³ carbons belonging to the γ-hydroxy-γ,γ-dimethylpropyl group and two sugar residues, the remaining 15 sp² carbons including 1 carbonyl group, 9 quaternary carbons (containing 5 oxygenated ones), and 5 methines, suggested that 1 possessed a flavonol skeleton.

Careful comparison of the NMR data with those of ikarisoside B (5) [14] showed high structural similarity except that the isoprenyl group in ikarisoside B was replaced a by γ-hydroxy-γ,γ-dimethylpropyl moiety in 1. The γ-hydroxy-γ,γ-dimethylpropyl moiety was deduced to be located at C-8 (δ_C = 107.9 ppm) according to heteronuclear multiple bond correlation (HMBC) correlations (Fig. 2) of H-11 with C-7 (δ_C = 163.0), C-8, and C-9 (δ_C = 155.7 ppm) and of H-12 with C-8. In addition, two sugar residues were identified as d-glucose (Glc) and l-rhamnose (Rha) by acid hydrolysis and gas chromatography (GC) analysis of their corresponding trimethylsilylated l-cysteine derivatives. The β-pyranosyl configuration of the glycosidic bond of Glc moiety was deduced from the coupling constant (J = 7.6 Hz) of its anomeric proton. Simultaneously, the anomeric configuration of Rha moiety was determined as the α-position according to ¹³C NMR chemical shifts (Table 2) [24]. The Rha moiety was located at C-3 (δ_C = 136.0 ppm) of the aglycone according to the HMBC correlation from H-1 (δ_H = 5.75 ppm) of Rha to C-3, whereas the Glc unit was linked to C-2 (δ_C = 82.7 ppm) of the Rha moiety due to the HMBC correlations from H-1 (δ_H = 4.43 ppm) of Glc to C-2 of Rha, as well as from H-2 (δ_H = 4.30 ppm) of Rha to C-1 (δ_C = 107.1 ppm) of Glc. Thus, the structure of 1 was unambiguously deduced as 8-γ-hydroxy-γ,γ-dimethylpropyl-5,7,4′-trihydroxy-flavonol-3-O-β-d-glucopyranosyl (1 → 2)-α-l-rhamnopyranoside and named as epimedkoreside A.

Fig. 2:

Key HMBC correlations of compounds 1 and 3.

Compound 2 was isolated as a yellow amorphous powder, and the molecular formula C₃₂H₄₀O₁₅ was deduced from the pseudomolecular ion [M + Na]⁺ at m/z = 687 in ESI-MS and further confirmed by the positive HR-ESI-MS (m/z = 687.2256 [M + Na]⁺), requiring 13 degrees of unsaturation. Detailed analysis of the ¹H and ¹³C NMR spectral data of 2 (Tables 1 and 2) and 1 made it clear that these two compounds were extremely similar except for the exchange of one monosaccharide. The protons at δ_H = 5.53 (s)/0.93 (d, J = 6.1 Hz) and 5.00 (s)/1.22 ppm (d, J = 6.2 Hz) in the ¹H NMR spectrum (Table 2) indicated the existence of two rhamnose residues (Rha and Rha′), which was further confirmed by hydrolysis and GC analysis. Moreover, one rhamnose moiety (Rha) was located at C-3 (δ_C = 136.1 ppm) of the aglycone according to the HMBC correlation from H-1 (δ_H = 5.53 ppm) of Rha to C-3. The other rhamnose moiety (Rha′) was located at C-2 (δ_C = 79.1 ppm) of the inner one (Rha) due to the HMBC correlations from H-1 (δ_H = 5.00 ppm) of Rha′ to C-2 of Rha and from H-2 (δ_H = 4.31 ppm) of Rha to C-1 (δ_C = 103.9 ppm) of Rha′. Based on the above evidence, compound 2 was unequivocally identified as 8-γ-hydroxy-γ,γ-dimethylpropyl-5,7,4′-trihydroxy-flavonol-3-O-α-l-rhamnopyranosyl(1 → 2)-α-l- rhamnopyranoside and named epimedkoreside B.

Compound 3, a yellow amorphous powder, was found to possess the molecular formula C₄₅H₆₀O₂₃ based on its HR-ESI-MS (m/z = 991.3409 [M + Na]⁺), in combination with ¹H and ¹³C NMR spectra. The ¹H NMR spectrum (Table 1) of 3 exhibited an O-methyl group at δ_H = 3.89 ppm (s), a singlet proton at δ_H = 6.62 ppm of a penta-substituted ring A, a pair of doublets at δ_H = 7.89 (J = 8.9 Hz), and 7.10 ppm (J = 8.9 Hz) of typical AA′BB′ system attributed to the protons of para-substituted ring B. In addition, a group of protons at δ_H = 3.53 (m, H_a-11), 3.62 (m, H_b-11), 5.17 (dd, J = 5.8 and 8.0 Hz, H-12), 1.63 (s, Me-14), and 1.71 ppm (s, Me-15), correlated with carbon signals at δ_C = 23.1 (t, C-11), 123.5 (d, C-12), 25.8 (q, C-14), and 18.4 ppm (q, C-15) in HSQC spectrum, respectively, suggesting the presence of a prenyl group [13, 14, 25]. Moreover, a series of signals at δ_H = 5.30 (d, J = 7.5 Hz), 5.56 (d, J = 1.3 Hz)/0.90 (d, J = 5.8 Hz), 4.99 (d, J = 1.3 Hz)/1.22 ppm (d, J = 6.2 Hz), as well as 5.34 (d, J = 1.3 Hz)/1.15 ppm (d, J = 6.2 Hz) (Table 2), revealed the presence of one glucose (Glc) and three rhamnose (Rha, Rha′, and Rha″) residues, in accordance with its acid hydrolysis and GC analysis results. As the NMR signals of four sugar units overlapped undesirably, the HSQC-TOCSY experiment was successfully used to distinguish and assign the ¹H and ¹³C NMR signals of each sugar unit. Besides the signals belonging to one methoxyl, one prenyl group, and four monosaccharides, the ¹³C NMR spectrum (Table 1) of 3 showed 15 sp² carbons (including 1 carbonyl, 9 quaternary ones, and 5 methines) for the flavonol skeleton.

The one- (1D) and two-dimensional (2D) NMR spectral data of 3 resembled those of acuminatoside (4) [13], except for one sugar moiety linked to C-7. The glycosidic linkages of 3 were determined by detailed analysis of HMBC spectrum (Fig. 2). A correlation of the proton at δ_H = 5.56 (d, J = 1.3 Hz, H-1 of Rha) to C-3 (δ_C = 136.5 ppm) of the aglycone indicated that the Rha moiety was attached to C-3. The Rha′ moiety was located at C-2 (δ_C = 78.9 ppm) of Rha due to the HMBC correlations from H-1 (δ_H = 4.99 ppm) of Rha′ to C-2 of Rha and from H-2 (δ_H = 4.28 ppm) of Rha to C-1 (δ_C = 103.7 ppm) of Rha″. The Glc unit was linked to C-7 (δ_C = 161.5 ppm) of the aglycone according to the HMBC correlation from H-1 (δ_H = 5.30 ppm) of Glc to C-7. The Rha″ moiety was connected with C-2 (δ_C = 80.2 ppm) of Glc based on the HMBC correlations from H-1 (δ_H = 5.34 ppm) of Rha″ to C-2 of Glc and from H-2 (δ_H = 3.73) of Glc to C-1 (δ_C = 102.3 ppm) of Rha″. Epimedkoreside C (3) was therefore elucidated as anhydroicaritin 3-O-α-l-rhamnopyranosyl(1 → 2)-α-l-rhamnopyranosyl-7-O-α-l-rhamnopyranosyl(1 → 2)β-d-glucopyranoside.

Compounds 5, 7, 9, and 12–15 were examined for their inhibition effect on superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (FMLP/CB)-induced human neutrophils to evaluate their anti-inflammatory potential. However, all compounds at 10-μm concentration exhibited inhibition lower than 50 %.

3 Experimental section